The association between maternal prenatal folic acid and multivitamin supplementation and autism spectrum disorders in offspring: An umbrella review

Biruk Beletew Abate; Biruk Shalmeno Tusa; Ashenafi Kibret Sendekie; Desie Temesgen; Kindie Mekuria; Addis Wondmagegn Alamaw; Molla Azmeraw; Alemu Birara Zemariam; Tegene Atamenta Kitaw; Amare Kassaw; Gebremeskel Kibret Abebe; Befkad Derese Tilahun; Gizachew Yilak; Molalign Aligaz Adisu; Berihun Dachew

doi:10.1371/journal.pone.0334852

. 2025 Nov 18;20(11):e0334852. doi: 10.1371/journal.pone.0334852

The association between maternal prenatal folic acid and multivitamin supplementation and autism spectrum disorders in offspring: An umbrella review

Biruk Beletew Abate ^1,^2,^*, Biruk Shalmeno Tusa ^1,³, Ashenafi Kibret Sendekie ^4,⁵, Desie Temesgen ⁶, Kindie Mekuria ⁶, Addis Wondmagegn Alamaw ⁶, Molla Azmeraw ⁶, Alemu Birara Zemariam ⁶, Tegene Atamenta Kitaw ⁶, Amare Kassaw ⁷, Gebremeskel Kibret Abebe ⁶, Befkad Derese Tilahun ⁶, Gizachew Yilak ⁶, Molalign Aligaz Adisu ⁶, Berihun Dachew ^1,⁸

Editor: Cecilia Nwadiuto Obasi⁹

¹School of Population Health, Curtin University, Perth, Washington Australia

²College of Medicine and Health Sciences, Woldia University, Woldia, Ethiopia

³Department of Epidemiology and Biostatistics, College of Health and Medical Sciences, Haramaya University, Haramaya, Ethiopia

⁴Department of Clinical Pharmacy, School of Pharmacy, College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia

⁵School of Pharmacy, Curtin Medical School, Faculty of Health Sciences, Curtin University, Bentley, Washington, Australia

⁶Department of Nursing, College of Health Science, Woldia University, Woldia, Ethiopia

⁷Department of Nursing, College of Health Science, Debre Tabor University, Ethiopia

⁸enAble Institute, Curtin University, Perth, Western Australia, Australia

⁹University of Port Harcourt, NIGERIA

Competing Interests: The authors have declared that no competing interests exist.

^✉

* E-mail: birukkelemb@gmail.com

Roles

Biruk Beletew Abate: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Biruk Shalmeno Tusa: Data curation, Formal analysis, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Ashenafi Kibret Sendekie: Data curation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Desie Temesgen: Conceptualization, Data curation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Kindie Mekuria: Conceptualization, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing

Addis Wondmagegn Alamaw: Conceptualization, Data curation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Molla Azmeraw: Conceptualization, Data curation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Alemu Birara Zemariam: Conceptualization, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Tegene Atamenta Kitaw: Conceptualization, Data curation, Validation, Visualization, Writing – original draft, Writing – review & editing

Amare Kassaw: Conceptualization, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Gebremeskel Kibret Abebe: Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing

Befkad Derese Tilahun: Conceptualization, Data curation, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Gizachew Yilak: Data curation, Investigation, Methodology, Supervision, Visualization, Writing – original draft, Writing – review & editing

Molalign Aligaz Adisu: Conceptualization, Data curation, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing

Berihun Dachew: Data curation, Formal analysis, Methodology, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Cecilia Nwadiuto Obasi: Editor

PMCID: PMC12626298 PMID: 41252376

Abstract

Background

Previous reviews have examined the association between maternal prenatal use of folic acid and multivitamin supplements and autism spectrum disorder (ASD) in children, but findings remain inconclusive. This umbrella review aims to synthesise the existing evidence on the association between prenatal folic acid and multivitamin supplementation and the risk of ASD in offspring.

Methods

This umbrella review followed the PRISMA guidelines to synthesise and report evidence from existing systematic reviews and meta-analyses (SRMs). Articles were searched in PubMed, Scopus, Web of Science, and Google Scholar. The quality of included studies was assessed using the Assessment of Multiple Systematic Reviews (AMSTAR). A weighted inverse variance random-effects model was applied to estimate pooled effects. The association was quantified using relative risks (RRs) with 95% confidence intervals (CIs). Subgroup analysis and sensitivity analysis were also conducted. Heterogeneity and publication bias were assessed.

Results

Eight SRMs comprising 101 primary studies and over three million mother-offspring pairs were included. Prenatal folic acid and/or multivitamin supplementation was associated with a 30% reduced risk of ASD in offspring (RR = 0.70, 95% CI: 0.62, 0.78; GRADE: highly suggestive). Subgroup analysis by supplement type showed that maternal prenatal multivitamin supplementation reduced the risk of ASD by 34% (RR = 0.66, 95% CI: 0.55–0.80; GRADE: highly suggestive), while folic acid supplementation was associated with a 30% reduction in ASD risk (RR = 0.70, 95% CI: 0.60–0.83; GRADE: highly suggestive).

Conclusion

Maternal prenatal folic acid and multivitamin supplementation are associated with a reduced risk of ASD in offspring. These findings have important public health implications, suggesting that prenatal supplementation could help mitigate the risk of ASD in children.

Background

Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder that has an impact on reciprocal social interactions, nonverbal communication, and understanding of social relationships [1]. It is frequently associated with co-occurring illnesses such as epilepsy, depression, anxiety, and attention deficit hyperactivity disorder, as well as problematic behaviours such as trouble sleeping and self-injury [1,2].

Autism spectrum disorder is a global public health problem affecting up to 1% of children around the world [3]. The etiology and risk factors have been proposed to a single or a group of genetic mutations and environmental influences [4]. Ongoing studies are most frequently environmental risk factors [5], and prenatal maternal nutrition are among the modifiable risk factors for ASD [6]. One of the maternal nutritional factors that may reduce the risk of offspring ASD is prenatal folic acid and multivitamin supplementation. Folic acid supports DNA methylation and epigenetic regulation, both critical for neurodevelopment, and aids in neural tube formation, protecting against defects associated with neurodevelopmental disorders [7,8]. Multivitamins, which typically contain essential nutrients like vitamin B12, vitamin D, and iodine, help maintain immune system balance, reduce inflammation, and support brain function by ensuring proper neurotransmitter synthesis and amino acid metabolism [9]. These nutrients contribute to optimal fetal brain development, potentially lowering the risk of ASD.

Early prevention is preferable to treatment, as no medication can address the core symptoms of ASD; available treatments focus on managing specific co-occurring conditions or symptoms. Maternal supplementation with folic acid and multivitamins before and during pregnancy represents one of the most accessible and cost-effective preventive strategies. To date, eight systematic reviews and meta-analyses (SRMs) comprising 101 primary studies have examined whether maternal prenatal folic acid supplementation reduces the risk of ASD in offspring. However, findings from previous reviews remain inconclusive. While some reviews have reported the association between prenatal folic acid and multivitamin supplementation and a lower risk of ASD, others have found no association. However, findings remain inconclusive due to several limitations in the existing literature, including variability in study design and population characteristics, differences in the definition and measurement of supplementation exposures (folic acid alone versus multivitamins, timing, dose, and duration), heterogeneity in ASD diagnostic methods (ranging from clinical diagnoses to parent-reported outcomes), and potential publication or reporting bias. These limitations have contributed to conflicting findings: while some SRMs report a protective effect of maternal folic acid and/or multivitamin supplementation [10–14], others find no significant association [15,16]. These inconsistencies underscore the need for a more comprehensive synthesis. An umbrella review, unlike a single SRM, offers a higher-level overview by integrating findings across multiple SRMs, critically appraising their methodological quality, and grading the overall strength of the evidence. Accordingly, this umbrella review aimed to synthesise these inconsistent findings to clarify the potential role of prenatal folic acid and multivitamin supplementation in reducing the risk of ASD in offspring.

Methods

Research design and searching strategy

This umbrella review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [17]. Additionally, to ensure the study’s rigor, we adhered to the PRIOR statement, a reporting guideline for overviews of reviews on healthcare interventions [18].

Articles were searched across four databases including PubMed, Scopus, Web of Science, and Google Scholar. Additionally, a complementary search was conducted in Google Scholar to capture grey literature sources. We performed a comprehensive systematic search for SRMs that assessed the association between prenatal folic acid and/or multivitamin supplementation and the risk of ASD on a global scale, using the PICO (Population, Intervention, Comparison, and Outcome) framework. The search strategy incorporated MeSH terms, keywords, and their combinations. Moreover, we manually reviewed references cited in relevant systematic reviews and conducted a snowball search to identify linked articles from these studies. The search terms included: (Preconception OR pre-conception OR prenatal) AND (Multivitamin OR vitamin OR mineral OR micronutrient OR antioxidant OR diet OR folic acid OR folic-acid) AND (autism OR autistic OR ASD OR Asperger OR pervasive developmental disorder OR PDD) AND (meta-analysis OR systematic review OR review).

Study selection/eligibility criteria

The retrieved SRMs were imported into EndNote 20 software. Screening followed a two-stage process: title and abstract screening, followed by full-text review. Two independent investigators (BB and MA) applied pre-specified inclusion criteria to identify relevant articles. Eligible studies met the following criteria: [i] had a defined literature search strategy, [ii] appraised the included studies using a relevant tool, and [iii] used a standard approach to pooling data and providing summary estimates. Studies were excluded if they lacked relevant outcome measures, were not in English, or were narrative reviews, editorials, correspondence, conference abstracts, or methodological studies. Disagreements in both title/abstract and full-text screening were addressed through structured discussions during consensus meetings, where team members collaboratively reviewed the criteria and reached a resolution based on predefined guidelines.

Criteria for considering studies for this umbrella review

Types of studies.

All SRMs-analyses assess the association between maternal prenatal multivitamins and/or folic acid supplementation and ASD in the offspring.

Types of participants.

Participants who have taken folic acid and/or multivitamin supplementation during the prenatal period, regardless of whether the pregnancy was single or multiple, and irrespective of their gestational age, were considered.

Types of interventions.

Folic acid and multivitamin supplementation were administered to women before pregnancy and compared with a placebo, no treatment, or an alternative agent to improve outcomes. We included studies where different regimens for helping folic acid and multivitamins were compared.

Outcomes of interest: The incidence of ASD among intervention groups (whose mothers have taken folic acid and multivitamins) compared to the control groups (whose mothers didn’t take folic acid and multivitamins) was estimated.

Setting: Global

Publication condition: Published articles that reported the effect of the outcome of interest

Quality assessment

The methodological quality of all included reviews was assessed by two independent reviewers using the Assessment of Multiple Systematic Reviews (AMSTAR) tool [19,20]. The quality was scored out of 11, with scores < 3, 4–7, and 8–11 indicating low, moderate, and high qualities, respectively. The decision as to whether to include a review was made based on meeting a pre-determined proportion of all criteria, or on certain criteria being met. Decisions about a scoring system or any cut-off for exclusion were made in advance and agreed upon by all reviewers before critical appraisal commences.

Data extraction

Data from the included SRM studies were extracted using a standardized data abstraction form, developed in an Excel spreadsheet. For each SRM study, the following data were extracted: (a) identification data (first author’s last name and publication year), (b) Review aim and type, (c) measure of association (odds ratio or relative risk with 95% confidence intervals), (d) number of primary studies included within each SRM study and their respective design type, (e total number of sample size included, (f) publication bias assessment methods and scores, (g) quality assessment methods and scores, (h) and data synthesis methods (random or fixed-effects model) [Table 1].

Table 1. Characteristics of included studies.

Sr No	Authors	Year	Study design	Number of articles included	Sample size	Supplement	Timing	Estimate (95%CI)
1.	Guo B-Q, et al.[10]	2019	SRM	5	231,163	Multivitamin	Prenatal	RR = 0.62 (0.45, 0.86)
2.	Guo B-Q, et al. [15]	2019	SRM	8	840,776	Folic acid	Prenatal	OR = 0.91 (0.73,1.13)
3.	Li M, et al.[11]	2019	SRM	20	1038013	Multivitamin	Prenatal	RR = 0.64 (0.46, 0.9)
4.	Friel C, et al.[16]	2021	SRM	10	904,947	Multivitamin	Prenatal	RR = 0.74 (0.53, 1.04)
5.	Liu X, et al. [12]	2021	SRM	10	9795	Folic acid	Prenatal	OR = 0.57 (0.41, 0.78)
6.	Wang M, et al.[27]	2017	SRM	16	4514	Folic acid	Prenatal	OR = 0.77 (0.64,0.93)
7.	Iglesias VL et al [13]	2018	SRM	16	756,365	Folic acid	Prenatal	OR = 0.58 (0.46, 0.75)
8.	Chen H et al.[14]	2023	SRM	15	1,116,337	Folic acid	Prenatal	OR = 0.68 (0.53,0.83)

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Statistical analysis

STATA v17.0 software was used for statistical analyses. Both narrative and qualitative methods, including tables and forest plots, were employed to summarize the estimates from the included reviews. We conducted an inverse variance-weighted random-effects meta-analysis to estimate pooled effect sizes with 95% CIs [21]. We assessed heterogeneity across studies using Cochrane’s Q test (Chi-square), the I² statistic, tau-squared (τ²), and corresponding p-values [22]. Heterogeneity was interpreted according to conventional thresholds, with I² values of 0% indicating no heterogeneity and values of 25%, 50%, and 75% representing low, moderate, and high heterogeneity, respectively [20,23]. For analyses where significant heterogeneity was detected, we applied the DerSimonian–Laird random-effects model. Subgroup analyses were conducted according to supplement type (folic acid versus multivitamin). To evaluate the robustness of findings, sensitivity analyses were performed by sequentially excluding individual studies. Publication bias was assessed using both graphical and statistical approaches: visual inspection of funnel plot symmetry and Egger’s regression test. Funnel plot asymmetry and a p-value <0.05 in Egger’s test were considered indicative of potential publication bias [24].

Credibility/GRADE assessment of each pooled analysis

We used the GRADE system to assess the strength of evidence for each pooled analysis, classifying it as convincing, highly suggestive, suggestive, or weak. GRADE uses five criteria: risk of bias, inconsistency, indirectness, imprecision, and publication bias. Downgrades occur based on a high risk of bias, inconsistency (high I² values), indirectness (multiple control comparisons), imprecision (wide confidence intervals or small sample size), and publication bias (significant Egger test p-value). Convincing evidence (Class I) required a p-value < 10 ⁻ ⁶, over 1,000 participants, low-to-moderate heterogeneity (I² < 50%), a 95% prediction interval excluding the null, and no small-study bias. Highly suggestive associations (Class II) involved over 1,000 participants, a p-value < 10 ⁻ ⁶, and a 95% prediction interval excluding the null. Suggestive evidence (Class III) required over 1,000 participants and a p-value ≤ 0.001, while weak associations (Class IV) needed a p-value ≤ 0.05, with p > 0.05 considered non-significant [25,26].

Ethics approval and consent to participate

This study is a systematic review of published articles and does not require ethical approval.

Results

A total of 1388 reviews were identified; 1380 were from different databases, and eight were from manual sources. After duplicates were removed, 667 articles remained to be screened for title and abstract. After title and abstract screening, 142 studies remained for full-text review. Finally, after excluding 134 (using eligibility criteria), 8 SRMs, comprising 101 primary studies with 3,029,208 participants were included in the final analysis (Fig 1).

Characteristics of included studies

All the included reviews were both systematic reviews and meta-analyses [10–16,27]. Five of the included SRMs examined the effect of maternal folic acid supplementation [12–15,27], while the other three investigated the impact of multivitamin supplementation [10,11,16] on ASD in offspring. The included SRMs range from five [14] to 20 [15] primary studies. The minimum and maximum sample sizes in these included SRM were 4,514 [27] and 1,090,585 [14] respectively (Table 1).

Quality assessment

Based on AMSTAR-2 criteria, the majority of the SRMs (6 out of 8) [10,12,13,15,16,27] were assessed as high quality, while 2 out of 8 were rated as low quality [11,14] (S1 Table). The most common reasons for a low-quality rating based on the AMSTAR-2 checklist are: failure to register the protocol in PROSPERO [10–13,15,27], absence of an evaluation of the risk of bias in the meta-analysis [10,11,13–15,27], insufficient explanation of heterogeneity [11,16], and failure to assess publication bias [10,11] (S1 Table).

Effect of maternal prenatal folic acid and multivitamin supplementation on offspring ASD

Among the eight studies included, six reviews reported an association between prenatal folic acid and multivitamin supplementation and a reduced risk of ASD [10–14], whereas the remaining two reviews, one for folic acid and one for multivitamin, found no such association [15,16]. The effect estimates from these included studies ranges from 0.57 (95% CI: 0.41, 0.78) [12] to 0.91 (95% CI: 0.73, 1.13) [10].The random-effects model analysis revealed a 30% reduced risk of ASD (RR = 0.70, 95% CI: 0.62–0.78) in offspring of mothers who took folic acid and/or multivitamin supplementation during prenatal care, compared to those who did not. Subgroup analysis by supplement type showed that maternal prenatal multivitamin supplementation reduced the risk of ASD by 34% (RR = 0.66, 95% CI: 0.55–0.80; GRADE: highly suggestive), while folic acid supplementation was associated with a 30% reduction in ASD risk (RR = 0.70, 95% CI: 0.60–0.83; GRADE: highly suggestive) (Fig 2).

Sensitivity analysis

The sensitivity analysis indicated that any of the single studies did not influence the pooled result. The pooled sensitivity estimate for the association between maternal prenatal folic acid and multivitamin supplementation and offspring ASD ranged from 0.67 (95% CI: 0.61–0.74) [15] to 0.72 (95% CI: 0.64–0.81) [13] (S1 Fig).

Publication bias assessment

The symmetrical funnel plots and Egger’s test provided no evidence of substantial publication bias among the included studies (P-value = 0.14).

Credibility according to GRADE evidence assessment

Overall, the strength of evidence assessed using the GRADE framework indicates highly suggestive (Class II) evidence for both the pooled analysis of the effects of maternal prenatal folic acid and multivitamin supplementation in preventing ASD. Both pooled analyses met most of the GRADE criteria. Specifically, both analyses demonstrated low to moderate heterogeneity, with I² values of 0.00% (P = 0.74) and 60.76% (P = 0.04), respectively. No publication bias was detected in either analysis (Egger’s test, P = 0.09). Furthermore, both analyses met the other three GRADE criteria: 1) no serious indirectness, 2) no serious imprecision, and 3) low risk of bias assessment using AMSTAR (A Measurement Tool to Assess Systematic Reviews), where 75% of questions were answered ‘yes’ and 25% were rated as ‘unclear’ or ‘no.’ As a result, the overall certainty of the evidence for both folic acid and multivitamin supplementation in preventing ASD highly suggests a 30% reduction in the risk of ASD for folic acid supplementation and a 34% reduction in the risk of ASD for multivitamin supplementation (S2 Table).

Discussion

In this umbrella review, we pooled the data from eight SRMs, and the results of the overall analysis indicated a lower risk of ASD among children whose mothers took prenatal folic acid and multivitamin supplements, compared to those children whose mothers did not take the supplements.

Our finding suggests that prenatal folic acid and multivitamin supplementation may play a protective role in reducing the risk of ASD in offspring. Several potential mechanisms could explain this association. Folic acid, a vital nutrient during pregnancy, is involved in DNA methylation and neural tube development, which are crucial for proper brain development [28–30]. Adequate folate levels during early pregnancy may help prevent neural defects that could contribute to neurodevelopmental disorders including ASD. Several existing studies have reported a link between reduced folate levels and an increased risk of neural tube defects [31–33]. Additionally, folic acid may play a role in modulating inflammation and oxidative stress [34], both of which have been implicated in the development of ASD. During neurogenesis and cell migration, high concentrations of methyl donors are required in most cortical and subcortical structures [35]. Studies have examined the possible causative relationship between folate intake during preconception and pregnancy its metabolism, and the onset of ASDs, given the role folate plays in the developing brain through DNA synthesis, neurotransmitter production, and myelination [10–16,27]. However, the results remain conflicting. The timing of maternal folate intake appears to be a critical factor; a child’s risk of autism was reduced only when the supplements were taken preconceptionally and during early pregnancy, specifically between 4 weeks before and eight weeks after the start of pregnancy [36].

The current review explored maternal exposure to multivitamins and folic acid supplementation during pregnancy and its association with ASD in offspring. However, identifying the specific ingredient(s) responsible for this protective effect remains challenging, as most studies did not provide detailed information on the composition or quantities of individual vitamins and minerals within the multivitamins used. Nutrients are vital environmental factors that play a crucial role in embryogenesis, fetal growth, and neurobiological development [37]. Multivitamins are among the most common dietary supplements, typically containing a combination of essential vitamins and minerals [38]. Among these, vitamin B9 (folate) is well-documented for its critical roles in neuronal differentiation, maturation, neurotrophic functions, and neuroprotection, alongside its traditional function in calcium and phosphorus metabolism. These attributes suggest folate may contribute significantly to the observed protective effects. Additionally, other studies have suggested that certain nutrients, such as zinc and vitamin D, may also play a role in the nutritional management of autism [39,40]. Vitamin D, similar to folate, is essential for neuronal differentiation, maturation, neurotrophic functions, and neuroprotection [41]. Beyond these roles, it is well known for its involvement in calcium and phosphorus regulation [42]. Zinc, although less discussed in the context of multivitamin supplementation, is recognized for its importance in brain development and function, making it a potential candidate for further exploration [40].

Folic acid deficiency before and during early pregnancy is a well-established risk factor for NTDs and may contribute to brain abnormalities associated with ASD [43–46]. Periconceptional folic acid supplementation has been shown to reduce NTD risk by up to 70% [47]. While a daily dose of 400 μg of folic acid is recommended to prevent NTDs, its role in reducing ASD risk remains complex and influenced by other factors [12]. Although emerging evidence suggests maternal folic acid supplementation may lower ASD risk, further rigorous population-based studies are needed to identify the most effective timing and dosage [47]. Understanding these factors is crucial for developing targeted strategies to prevent neurodevelopmental disorders.

Strengths and limitations of the study

Several limitations should be acknowledged. First, the included systematic reviews and meta-analyses did not cover all global regions, which may limit the generalizability of the findings. Second, mild to moderate heterogeneity persisted despite efforts to address it through subgroup analyses and random-effects modeling. Third, variations in supplementation timing, dosage, and formulation across studies posed challenges for interpretation. Finally, some reviews lacked protocol registration and did not consistently assess risk of bias or publication bias, potentially affecting the reliability of their conclusions.

Despite these limitations, this umbrella review advances the literature by jointly analysing both folic acid and multivitamin supplementation, providing a more comprehensive synthesis of their potential impact on ASD risk. The study adhered to PRISMA guidelines, ensuring methodological rigor, and evaluated the quality of included reviews using the AMSTAR tool [48–51]. Subgroup analyses by supplement type further enhanced the robustness of the findings.

Implications for practice, policy, and future research

Practice: The findings emphasize the importance of prenatal folic acid and multivitamin supplementation as part of routine maternal care to reduce the risk of ASDs in offspring. Healthcare providers should encourage women to start supplementation during the pre-conception period and continue throughout pregnancy. This proactive approach can be integrated into preconception and prenatal care programs to promote maternal and fetal health.

Policy: Policymakers should prioritize the inclusion of folic acid and multivitamin supplementation in national maternal health guidelines and public health initiatives. Public awareness campaigns are needed to educate women of reproductive age about the potential benefits of these supplements in reducing ASD risk. Subsidizing or ensuring the affordability of high-quality folic acid and multivitamin supplements, particularly in low-resource settings, can further enhance accessibility and adherence to these recommendations.

Research: Future research should focus on conducting large-scale prospective cohort studies and randomized controlled trials (RCTs) to confirm and expand these findings. Key areas of investigation include exploring the molecular mechanisms behind the protective effects of folic acid and multivitamins against ASD, determining the optimal dosage, duration, and timing of maternal supplementation for maximum risk reduction, and assessing the roles of specific nutrients like vitamin D, zinc, and folate in mitigating ASD risk. Additionally, it is important to evaluate the impact of maternal supplementation across diverse populations and geographic regions to enhance generalizability and ensure culturally relevant interventions. Addressing these gaps will strengthen evidence to refine clinical guidelines and improve preventive strategies for ASD.

Conclusion and recommendation

This umbrella review found that maternal prenatal folic acid or multivitamin supplementation was associated with a reduced risk of ASD in children. The evidence is highly suggestive, as assessed using the GRADE approach, indicating strong support for the protective effects of these supplements. Incorporating prenatal multivitamin and folic acid supplementation into ASD prevention strategies is recommended during the pre-conception and early pregnancy period. Further large-scale studies and RCTs are needed to clarify their protective effects, underlying mechanisms, and optimal dosage, timing, and duration for reducing ASD risk.

Supporting information

S1 Fig. Sensitivity analysis for the effect of pre-conception FA and multivitamins for the prevention of autism.

(TIF)

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S2 Fig. Funnel plot for the effect of pre-conception FA and multivitamins for the prevention of autism.

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S1 Table. Search strategy used for one of the databases.

(DOCX)

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S2 Table. Shows quality appraisal of included SRM using AMSTAR-2 checklist.

(DOCX)

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S3 Table. Quality assessment using the GRADE framework of each pooled analysis.

(DOCX)

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S4 Table. PRISMA_2020_checklist.

(DOCX)

pone.0334852.s006.docx^{(32.4KB, docx)}

S5 Table. Descriptions of 667 articles.

(XLSX)

pone.0334852.s007.xlsx^{(74.6KB, xlsx)}

Data Availability

Data are all contained within the paper and/or Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0334852.r001

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6 Jan 2025

Dear Dr. Abate,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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comments/suggestion to author; In order to published this manuscript, you have to dress the two reviewers comments one by one. The two reviewers addressed and critical reviewed the manuscript so that there is no conflict between them. Finally I need changes in this manuscript, grammatically, throughout the manuscript, and in the discussion part, you discussed only folic acid effects but no multivitamins .

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Reviewer #1: Pre-conception folic acid and multivitamin supplementation for prevention of autism in offspring: An umbrella review of Systematic Review and Meta-analysis

Thank you for the opportunity to review this umbrella review. It addresses an important question, however it is not effective in the message it endeavors to deliver. There are some major grammatical and formatting errors throughout, specifically the results tables. I am not convinced that this manuscript adds to what is already known regarding supplementation and outcomes of ASD. I believe there could be more substantial analysis, and a greater discussion regarding the results and the implications.

Abstract

Result : please explain SRM.

‘Subgroup analysis by type of supplement revealed that the pooled effect of either folic acid or multivitamin supplementation for the prevention of autism was found to be 0.69(0.58, 0.80) in the multivitamin group, while this estimate is 0.70(0.55,0.85) among studies with folic acid supplementation.’ Please give units/meaning to this figures, and rephrase this to avoid such repetition, for example Subgroup analysis by type of supplement revealed that the pooled effect of either folic acid alone or multivitamin supplementation for the prevention of autism was found to be 0.70(0.55,0.85) and 0.69(0.58, 0.80) respectively.

‘with a slightly higher (1%) percentage of reduction in autism’ please rephrase to: with a 1% greater reduction in autism’.

Introduction

‘Ongoing studies are most frequently considered in environmental risk factors’ can you reference this please?

‘Preconception and prenatal maternal nutrition are among the modifiable risk factors of ASD’ please add a reference.

‘The current review explored that during pregnancy, maternal exposure to Multivitamins and folic acid supplementation significantly reduces the likelihood of ASD in offspring.’ Please rephrase – the current review explores maternal exposure to multivitamins and folic acid supplementation during pregnancy and its association with ASD in offspring’ or something similar.

‘Folic is water soluble’ please add folic acid.

‘Various folate forms participate in essential reactions, like DNA methylation and replication’ – needs a reference.

‘FA is metabolized differently to naturally occurring forms of folates with a bioavailability of approximately 70% higher. 7’ Please also rephrase this sentence as it is not clear what the meaning of it is.

Folic acid is not abbreviated to FA prior to this – please use folic acid (FA) at first use, and then continue to use FA throughout. Please be consistent in either using the full term of folic acid or the abbreviation FA.

‘Since folate is essential for metabolism and nervous system function and as a precursor of S-adenosyl-methionine (SAM), the universal methyl donor that is essential for cellular methylation of lipids, proteins, nucleic acid, and metabolites, inadequate availability affects the gene expression in the neurodevelopmental process which may have an association between maternal folate level during pregnancy and ASD mediated by DNA methylation. 8, 9’ please shorten this sentence or break it into multiple sentences. It is quite long as it is and difficult to follow for the reader.

Methods

SRM is introduced without full explanation – please write the term in full before using it as an abbreviation.

‘From checking PROSPERO, this umbrella review needed to be registered.’ Please remove.

‘We searched systematic review and meta-analysis’ please rephrase to We searched for systematic reviews and meta-analyses

‘in global context on 23/10/ 2023 G. C. Were searched SRM which reported the effect of preconception folic acid and/or multivitamin supplementation for prevention of autism in global context using PICO frameworks’ please rephrase as this is incorrect grammar and difficult to decipher what you are saying, specifically at the mention of G.C. and we ‘searched SRM which reported…’

PICO only needs to be mentioned once.

‘was also performed’ remove also.

‘The quality scoring was done out of 11, with scores 8–11, 4–7, and < 3 indicating high, medium, and low qualities’ Please re-organize this sentence to start from low, medium and high so that it reads chronologically.

There is only mention of investigators screening the title and abstracts, what about the full text screening? How were disagreements resolved?

‘After the data was extracted using Microsoft Excel format, we imported the data to STATA version 17.0 statistical software for further analysis’ please reduce this to ‘STATA v17.0 was used for statistical analyses.

Results

‘After duplication was removed’ please correct the grammar so that it reads ‘ after duplicates were removed’.

The flow chart needs clarification – Were the 721 duplicates? Or that’s how many remained after duplicate removal? It needs to be cleaned in terms of layout also – the boxes are not in line, the arrows are not in line, the text in the blue box doesn’t fit. It needs to be cleaned.

Please highlight further the ‘quality reasons’ for exclusion in your methods.

‘Included SRM included from five to 16 studies’ please rephrase to avoid repetition of included, it is confusing.

‘….0.57, 95% CI: 0.41, 0.78) 15 to 0.91, 95% CI: 0.73, 1.13)’ please fix the use of brackets.

‘The random-effects model analysis from those studies revealed that, the pooled effect of either folic acid or multivitamin supplementation’ remove the comma or rephrase please.

‘This indicates prenatal folic acid and /or folic acid supplementation was associated’ please correct this – there is no distinction between the and/or folic acid mentioned.

‘Subgroup analysis was done through stratified by the type of supplementation. Based on this..’ please rephrase. Remove ‘Based on this’.

‘Based on this, the pooled effect of either folic acid or multivitamin supplementation for the prevention of autism was found to be 0.69(0.58, 0.80) in in multivitamin group, while this estimate is 0.70(0.55,0.85) among studies with folic acid supplementation’ remove the second ‘in’ and rephrase so that each supplementation type is only mentioned once – there is too much repetition. Please give meaning to the figures – there is no unit or indication as to what these figures represent.

‘which indicated that, the absence of publication bias’ rephrase to ‘which indicates the absence of publication bias’.

It is evident from your results that you are using screenshots from the STATA output of results. Please enter these into appropriately formatted tables, only highlighting the necessary information as opposed to the excess of information and the many decimal points from the screenshot. It is inappropriate.

‘Our pooled estimated effect of preconception folic acid and multivitamin for the prevention of autism varied from 0.68 (0.57– 0.78) to 0.71 (0.62–0.81) after the deletion of a single study (Fig 5).’ Use the full-term figure as opposed to fig, and give meaning to the numbers.

I think it would be interesting to ascertain if the multivitamins used in the studies contained folic acid. The introduction highlighted the lack of consensus regarding dose, frequency and timing of supplementation, but this was not addressed in the results.

There is little mentioned regarding the quality of the reviews included in the results. Please rectify.

As this is a global context, I think it would be interesting to see exactly what countries were included. I would imagine a difference would occur depending on socioeconomic status of the countries included, among other environmental factors.

Discussion

‘increases the risk of (NTDs)’ please include Neural Tube Defects.

‘insufficient folic acid intake during the early stages of pregnancy can result in (NTDs),’ remove brackets around NTDs.

Your discussion is brief, and focuses only on folic acid, when the review includes multivitamins. Please consider the numerous aspects at play here, the discussion to me does not consider all of the evidence and is very one-sided. Please broaden.

Is 7 studies considered substantial? Particularly when you started with >1000. I would have noted the low number of studies included.

You mention AMSTAR is used but there is no evidence of this use in your results.

‘Despite these strengths the study also has few limitations: as the included studies were not from all countries and this may affect the generalizability of the pooled result’ this should be rephrased to improve grammar.

‘despite the authors tried to reduce it through’ please rephrase to ‘despite the authors efforts to reduce it…’

‘Participants who took multivitamin supplementation were associated with a slightly higher (1%) percentage of reduction in autism compared with those who took folic acid.’ Rephrase as highlighted previously.

I am not convinced that this umbrella review really adds to what is already known. Please elaborate in your discussion, highlighting the importance of this work and how it addresses the gaps in our existing knowledge. I feel as though the introduction leads the reader to believe that this will look at importance dose quantity, frequency, supplementation timing, etc., which is not the case.

Reviewer #2: Abstract

IMPORTANCE: Previous reviews explored the association between maternal use of folic acid and multivitamin supplements and the risk of autism spectrum disorders (ASD) in

OBJECTIVE: To combine the inconsistent data on the effect of prenatal folic acid and multivitamin supplementation for the prevention of autism in offspring.

DESIGN, SETTING, AND PARTICIPANTS: In this umbrella review using PRISMA guideline, PubMed, Embase, Scopus, Web of Sciences, Cochrane Database of Systematic Reviews, Scopus, and Google Scholar which reported the effects of folic acid and multivitamin supplementation for the prevention of autism in offspring were searched. The quality of the included studies was assessed using the Assessment of Multiple Systematic Reviews (AMSTAR). A weighted inverse variance random-effects model was applied to find the pooled estimates. The subgroup analysis, heterogeneity, publication bias, and sensitivity analysis were also assessed.

EXPOSURES: Maternal multivitamin/folic supplements were classified for folic acid, multivitamin supplements, and combination thereof exposed in the intervals before pregnancy. MAIN OUTCOMES AND MEASURES: The association between maternal multivitamin supplementation and the risk of autism in offspring was quantified with relative risks (RRs) and their 95% Cis.

RESULT: Seven SRMs with 3,785,573 study participants were included. The pooled effect of either folic acid or multivitamin supplementation for the prevention of autism globally is found to be 0.69 (95% CI: 0.60, 0.79) (I2 = 46.4%; p <=0•083). Subgroup analysis by type of supplement revealed that the pooled effect of either folic acid or multivitamin supplementation for the prevention of autism was found to be 0.69(0.58, 0.80) in the multivitamin group, while this estimate is 0.70(0.55,0.85) among studies with folic acid supplementation.

CONCLUSION AND RELEVANCE: This umbrella review of systematic review and meta analysis found that prenatal folic acid and multivitamin supplementation was associated with a 31% reduction in autism. Participants who took multivitamin supplementation were associated with a slightly higher (1%) percentage of reduction in autism compared with those who took folic acid. This comprehensive umbrella review revealed both folic acid and multivitamins were associated with significantly lower levels of autism in children. Considering the incorporation of those supplements in autism prevention strategies during the pre-conception period is recommended. More large-scale prospective cohorts and RCTs are needed to understand the protective effect of multivitamins/ and or folic acid on ASD risk, address the molecular mechanisms, and determine the optimal dose, duration, and timing of maternal multivitamin/ and folic acid intake for best child ASD risk reduction.

I do have some comments in the abstract section

� you should state introduction other than importance,

� DESIGN, SETTING, AND PARTICIPANTS these are already methods

� Once you stated the number in result section so you should have to state general context of your finding in conclusion for example you got 69%( result) your conclusion might be (high or low based specific guideline to say high or low)

Once in your result section you got prenatal folic acid and multivitamin supplementation for the prevention of autism globally is found to be 0.69, and in the conclusion prenatal folic acid and multivitamin supplementation was associated with a 31% reduction in autism. Am confusing with these what does it mean the finding 69% and 31% in conclusion, you see both sentences revealed reduction of autism.

Does it prisma guideline a search engine?

Key Points QUESTION: Does maternal folic acid and multivitamin supplement use before pregnancy decrease the risk of autism in offspring?

FINDINGS In this umbrella review, seven systematic reviews and meta-analyses were included with 3,785,573 participants. A statistically significant association between maternal multivitamin and folic acid supplement use before pregnancy and reduced risk of autism in their offspring was observed. MEANING A reduced risk of autism spectrum disorder in children born to women who used multivitamins and folic acid supplements before pregnancy has important public health implications; possible mechanisms include epigenetic modifications.

Is that seven systematic review with 3,785,573 participant your key findings? It is better to report the main findings that you got from 7 systematic review articles

However, evidence derived from the previous reviews on the uses of folic acid and multivitamins for the prevention of ASD is inconclusive. Some studies concluded that there is no association between maternal folic acid supplementation during the prenatal period and the risk of ASD. 10, 11 On the other hand, other reviews revealed that there is a strong association between preconception and prenatal folic acid and multivitamin supplementations and the risk of autistic disorder. 12-15 Even for those who agree on the supplementations, there are inconsistencies in the timing (preconception or during pregnancy), the types of Nutrients (folic and Multivitamins), and each nutrient's dose. Therefore, this Umbrella review aims to explore the actual association between maternal supplementations with folic acid and multivitamins pre-conceptional and during pregnancy and the risk of ASD in offspring.

Your interest is to do umbrella review, it is not elaborated more what is the important to do umbrella review, you only said “previous reviews on the uses of folic acid and multivitamins for the prevention of ASD is inconclusive.” What are the key features of umbrella review? What gaps were not answered by systematic review? You should have to clearly show the need to use umbrella review in the context of your title

All published, unpublished, and systematic reviews and meta-analyses assess the effect of pre-conceptional folic acid and multivitamin supplementation for preventing autism in the offspring. We would have included studies reported as abstracts only and those with full-text manuscripts

We searched systematic review and meta-analysis from the following databases: PubMed, Embase, Scopus, Web of Sciences, Cochrane Database of Systematic Reviews, Scopus and Google Scholar in global context on 23/10/ 2023 G. C.

Potentially eligible studies identified through database search (n=1380), Potentially eligible studies identified through other sources (n =8)

How many articles have you identified through different search engine ( i.e how much was in Pub med, scops, unpublished article…etc )

Results and Discussion:

I did not see the implications of the study for policymakers and practitioners. Please provide unique, actionable strategies to address autism based on the findings.

Ensure that the discussion goes beyond restating the results and connects them to broader public health and policy contexts.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: Yes: Belayneh Jejaw Abate

**********

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PLoS One. 2025 Nov 18;20(11):e0334852. doi: 10.1371/journal.pone.0334852.r002

Author response to Decision Letter 1

1 May 2025

Dear Khem Descatamiento,,

Thank you for your thoughtful feedback.

Please find attached a document containing signatures from all authors, confirming our agreement with the proposed authorship changes as requested.

File name:Authorship Change PONE_all signed

Kind regards,

Biruk Beletew Abate

On behalf of all authors

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PLoS One. doi: 10.1371/journal.pone.0334852.r003

Decision Letter 1

Cecilia Nwadiuto Obasi

26 Sep 2025

Dear Dr. Abate,

Please submit your revised manuscript by Nov 10 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Kind regards,

Cecilia Nwadiuto Obasi, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

Reviewer #2: Thank you to the authors for their efforts in addressing the previous comments. That said, I have a major concern regarding the introduction section, summarized as follows:

1. Although you mention that previous findings are "inconclusive," it is important to specify why this is the case. What are the particular limitations or gaps in the existing literature that your umbrella review aims to address? Additionally, please clarify the added value of an umbrella review compared to a standard systematic review.

2. You note that some reviews report an association while others do not, but you do not discuss potential reasons for these conflicting results. Exploring these discrepancies is essential, as it forms the main rationale for conducting an umbrella review.

3. The introduction should also briefly explain what an umbrella review entails and why this approach is the most suitable method to resolve the current inconsistencies, rather than conducting another standard systematic review.

Reviewer #3: Based on the revised manuscript, the authors have made substantial improvements in both methodological clarity and narrative structure. The previously raised concerns related to the background length, clarity of methodological reporting, justification of model selection, and robustness of findings have now been comprehensively addressed.

Reviewer #4: In the Methods

Although the manuscript states that heterogeneity and publication bias were assessed, the methodology used to evaluate these aspects is not adequately described. Specifically, the authors should clarify which statistical tests or graphical methods were employed to assess heterogeneity, such as Cochrane’s Q test, I² statistic, or tau-squared, and explain how the degree of heterogeneity was interpreted. Similarly, for publication bias, it is important to specify whether funnel plots, Egger’s regression test, or other techniques were used, and how the results were interpreted.

DISCUSSION:

Better to adequately indicate the study's limitations and than statements related to strengths

This umbrella review stands apart from prior studies by jointly analyzing both folic acid and multivitamin supplementation, offering a broader synthesis of their potential impact on autism spectrum disorder (ASD) risk. However, several limitations should be noted. The included systematic reviews and meta-analyses do not represent all global regions, which may restrict the generalizability of the findings. Mild to moderate heterogeneity persists despite subgroup analyses and the use of random-effects modeling. Additionally, inconsistencies in supplementation timing, dosage, and formulation across studies introduce interpretive challenges. Some reviews lacked protocol registration and did not consistently assess risk of bias or publication bias, which may affect the reliability of their conclusions.

**********

what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #2: Yes: Belayneh Jejaw Abate

Reviewer #3: Yes: Pradeep Kumar

Reviewer #4: No

**********

PLoS One. 2025 Nov 18;20(11):e0334852. doi: 10.1371/journal.pone.0334852.r004

Author response to Decision Letter 2

1 Oct 2025

PONE-D-24-14480R1

The association between maternal prenatal folic acid and multivitamin supplementation and autism spectrum disorders in offspring: An umbrella review

PLOS ONE

Cecilia Nwadiuto Obasi, PhD

Academic Editor

PLOS ONE

Dear Dr. Cecilia Nwadiuto Obasi,

Thank you for the opportunity to submit a revised version of our manuscript titled " The association between maternal prenatal folic acid and multivitamin supplementation and autism spectrum disorders in offspring: An umbrella review”. We greatly appreciate the time and effort the reviewers have invested in providing feedback and their insightful comments and valuable suggestions. We have carefully addressed all concerns in a point-by-point manner and made the necessary revisions to the manuscript. These changes have been highlighted in yellow in the revised version for your reference. Below, you will find a detailed, point-by-point response to the reviewers’ comments and concerns. All page numbers refer to the clean, revised version of the manuscript.

Kind regards,

Biruk Abate

School of Population Health, Curtin University, Australia

Email: b.abate@postgrad.curtin.edu.au

Reviewer #2

Reviewer Comment: Thank you to the authors for their efforts in addressing the previous comments. That said, I have a major concern regarding the introduction section, summarized as follows: 1. Although you mention that previous findings are "inconclusive," it is important to specify why this is the case. What are the particular limitations or gaps in the existing literature that your umbrella review aims to address? Additionally, please clarify the added value of an umbrella review compared to a standard systematic review. 2. You note that some reviews report an association while others do not, but you do not discuss potential reasons for these conflicting results. Exploring these discrepancies is essential, as it forms the main rationale for conducting an umbrella review. 3. The introduction should also briefly explain what an umbrella review entails and why this approach is the most suitable method to resolve the current inconsistencies, rather than conducting another standard systematic review.

Authors’ Response: We sincerely thank the reviewer for the insightful comments. We have now revised the introduction accordingly and clarified the distinct contribution of our work. Specifically, an umbrella review systematically synthesises evidence from multiple SRMs, assesses their methodological quality, and evaluates the overall strength and consistency of the evidence. Unlike a single SRM, this higher-level synthesis integrates and appraises all available reviews, highlights both consistencies and discrepancies, and provides an overall grading of the evidence. This approach allows us to clarify whether maternal folic acid and/or multivitamin supplementation is consistently associated with a reduced risk of ASD in offspring.

We have revised the gap statement in the introduction section as follows: “Early prevention is preferable to treatment, as no medication can address the core symptoms of ASD; available treatments focus on managing specific co-occurring conditions or symptoms. Maternal supplementation with folic acid and multivitamins before and during pregnancy represents one of the most accessible and cost-effective preventive strategies. To date, seven systematic reviews and meta-analyses (SRMs) comprising 101 primary studies have examined whether maternal prenatal folic acid supplementation reduces the risk of ASD in offspring. However, findings from previous reviews remain inconclusive. While some reviews have reported the association between prenatal folic acid and multivitamin supplementation and a lower risk of ASD, others have found no association. However, findings remain inconclusive due to several limitations in the existing literature, including variability in study design and population characteristics, differences in the definition and measurement of supplementation exposures (folic acid alone versus multivitamins, timing, dose, and duration), heterogeneity in ASD diagnostic methods (ranging from clinical diagnoses to parent-reported outcomes), and potential publication or reporting bias. These limitations have contributed to conflicting findings: while some SRMs report a protective effect of maternal folic acid and/or multivitamin supplementation (10-14), others find no significant association (15, 16). These inconsistencies underscore the need for a more comprehensive synthesis. An umbrella review, unlike a single SRM, offers a higher-level overview by integrating findings across multiple SRMs, critically appraising their methodological quality, and grading the overall strength of the evidence. Accordingly, this umbrella review aimed to synthesise these inconsistent findings to clarify the potential role of prenatal folic acid and multivitamin supplementation in reducing the risk of ASD in offspring. (Page 3& 4: Line 72-93)”

Reviewer #3:

Reviewer Comment: Based on the revised manuscript, the authors have made substantial improvements in both methodological clarity and narrative structure. The previously raised concerns related to the background length, clarity of methodological reporting, justification of model selection, and robustness of findings have now been comprehensively addressed.

Authors’ Response: We sincerely thank Reviewer #3 for the constructive feedback, which has significantly improved the clarity and quality of the manuscript.

Reviewer #4

Reviewer Comment: Although the manuscript states that heterogeneity and publication bias were assessed, the methodology used to evaluate these aspects is not adequately described. Specifically, the authors should clarify which statistical tests or graphical methods were employed to assess heterogeneity, such as Cochrane’s Q test, I² statistic, or tau-squared, and explain how the degree of heterogeneity was interpreted. Similarly, for publication bias, it is important to specify whether funnel plots, Egger’s regression test, or other techniques were used, and how the results were interpreted.

Authors’ Response: We sincerely thank the reviewer for highlighting the need for greater clarity regarding our assessment of heterogeneity and publication bias. In response, we have now revised the methodology section to explicitly describe the statistical tests and graphical methods employed. Specifically, we used Cochrane’s Q test, the I² statistic, and tau-squared (τ²) to assess heterogeneity, and we interpreted the degree of heterogeneity following established thresholds (I² values of 25%, 50%, and 75% representing low, moderate, and high heterogeneity, respectively). For publication bias, we employed funnel plots and Egger’s regression test, with asymmetry in the funnel plot and a p-value <0.05 in Egger’s test considered suggestive of potential bias. We have also clarified how these results were interpreted in the context of our findings (See Methods, Page 7&8, Lines 164-175).

We have revised the gap statement in the Methods section as follows:

We assessed heterogeneity across studies using Cochrane’s Q test (Chi-square), the I² statistic, tau-squared (τ²), and corresponding p-values (22). Heterogeneity was interpreted according to conventional thresholds, with I² values of 0% indicating no heterogeneity and values of 25%, 50%, and 75% representing low, moderate, and high heterogeneity, respectively (20, 23). For analyses where significant heterogeneity was detected, we applied the DerSimonian–Laird random-effects model. Subgroup analyses were conducted according to supplement type (folic acid versus multivitamin). To evaluate the robustness of findings, sensitivity analyses were performed by sequentially excluding individual studies. Publication bias was assessed using both graphical and statistical approaches: visual inspection of funnel plot symmetry and Egger’s regression test. Funnel plot asymmetry and a p-value <0.05 in Egger’s test were considered indicative of potential publication bias (24).

DISCUSSION:

Reviewer Comment: Better to adequately indicate the study's limitations and then statements related to strengths. This umbrella review stands apart from prior studies by jointly analyzing both folic acid and multivitamin supplementation, offering a broader synthesis of their potential impact on autism spectrum disorder (ASD) risk. However, several limitations should be noted. The included systematic reviews and meta-analyses do not represent all global regions, which may restrict the generalizability of the findings. Mild to moderate heterogeneity persists despite subgroup analyses and the use of random-effects modeling. Additionally, inconsistencies in supplementation timing, dosage, and formulation across studies introduce interpretive challenges. Some reviews lacked protocol registration and did not consistently assess risk of bias or publication bias, which may affect the reliability of their conclusions.

Authors’ Response: We thank the reviewer for the insightful comments. We have now revised the discussion section accordingly (see discussion, Pages 12&13, Lines 296-308)

We have revised the limitation statements in the Discussion section as follows:

Strengths and Limitations of the Study

Despite these limitations, this umbrella review advances the literature by jointly analysing both folic acid and multivitamin supplementation, providing a more comprehensive synthesis of their potential impact on ASD risk. The study adhered to PRISMA guidelines, ensuring methodological rigor, and evaluated the quality of included reviews using the AMSTAR tool. Subgroup analyses by supplement type further enhanced the robustness of the findings.

PLoS One. doi: 10.1371/journal.pone.0334852.r005

Decision Letter 2

Cecilia Nwadiuto Obasi

3 Oct 2025

The association between maternal prenatal folic acid and multivitamin supplementation and autism spectrum disorders in offspring: An umbrella review

PONE-D-24-14480R2

Dear Dr. Abate,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Cecilia Nwadiuto Obasi, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0334852.r006

Acceptance letter

Cecilia Nwadiuto Obasi

PONE-D-24-14480R2

PLOS ONE

Dear Dr. Abate,

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Sensitivity analysis for the effect of pre-conception FA and multivitamins for the prevention of autism.

(TIF)

pone.0334852.s001.tif^{(144.8KB, tif)}

S2 Fig. Funnel plot for the effect of pre-conception FA and multivitamins for the prevention of autism.

(TIF)

pone.0334852.s002.tif^{(93KB, tif)}

S1 Table. Search strategy used for one of the databases.

(DOCX)

pone.0334852.s003.docx^{(15.5KB, docx)}

S2 Table. Shows quality appraisal of included SRM using AMSTAR-2 checklist.

(DOCX)

pone.0334852.s004.docx^{(20.8KB, docx)}

S3 Table. Quality assessment using the GRADE framework of each pooled analysis.

(DOCX)

pone.0334852.s005.docx^{(18.2KB, docx)}

S4 Table. PRISMA_2020_checklist.

(DOCX)

pone.0334852.s006.docx^{(32.4KB, docx)}

S5 Table. Descriptions of 667 articles.

(XLSX)

pone.0334852.s007.xlsx^{(74.6KB, xlsx)}

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Attachment

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pone.0334852.s010.docx^{(72.2KB, docx)}

Data Availability Statement

Data are all contained within the paper and/or Supporting Information files.

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PERMALINK

The association between maternal prenatal folic acid and multivitamin supplementation and autism spectrum disorders in offspring: An umbrella review

Biruk Beletew Abate

Biruk Shalmeno Tusa

Ashenafi Kibret Sendekie

Desie Temesgen

Kindie Mekuria

Addis Wondmagegn Alamaw

Molla Azmeraw

Alemu Birara Zemariam

Tegene Atamenta Kitaw

Amare Kassaw

Gebremeskel Kibret Abebe

Befkad Derese Tilahun

Gizachew Yilak

Molalign Aligaz Adisu

Berihun Dachew

Roles

Abstract

Background

Methods

Results

Conclusion

Background

Methods

Research design and searching strategy

Study selection/eligibility criteria

Criteria for considering studies for this umbrella review

Types of studies.

Types of participants.

Types of interventions.

Quality assessment

Data extraction

Table 1. Characteristics of included studies.

Statistical analysis

Credibility/GRADE assessment of each pooled analysis

Ethics approval and consent to participate

Results

Fig 1. PRISMA flow diagram shows the search results and reasons for the exclusion.

Characteristics of included studies

Quality assessment

Effect of maternal prenatal folic acid and multivitamin supplementation on offspring ASD

Fig 2. Forest plot shows the pooled effect of pre-conception FA and multivitamins for the prevention of autism.

Sensitivity analysis

Publication bias assessment

Credibility according to GRADE evidence assessment

Discussion

Strengths and limitations of the study

Implications for practice, policy, and future research

Conclusion and recommendation

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Engidaw Fentahun Enyew

Roles

Author response to Decision Letter 1

Decision Letter 1

Cecilia Nwadiuto Obasi

Roles

Author response to Decision Letter 2

Decision Letter 2

Cecilia Nwadiuto Obasi

Roles

Acceptance letter

Cecilia Nwadiuto Obasi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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